Biomedical subjects

Paul Matsudaira

Publications and source records attributed to Paul Matsudaira.

5 recordsLinked to original sources

PubMed2003

Macrophage podosomes assemble at the leading lamella by growth and fragmentation.

Podosomes are actin- and fimbrin-containing adhesions at the leading edge of macrophages. In cells transfected with beta-actin-ECFP and L-fimbrin-EYFP, quantitative four-dimensional microscopy of podosome assembly shows that new adhesions arise at the cell periphery by one of two mechanisms; de novo podosome assembly, or fission of a precursor podosome into daughter podosomes. The large podosome cluster precursor also appears to be an adhesion structure; it contains actin, fimbrin, integrin, and is in close apposition to the substratum. Microtubule inhibitors paclitaxel and demecolcine inhibit the turnover and polarized formation of podosomes, but not the turnover rate of actin in these structures. Because daughter podosomes and podosome cluster precursors are preferentially located at the leading edge, they may play a critical role in continually generating new sites of cell adhesion.

Actins↗

PubMed2003

The Saccharomyces cerevisiae calponin/transgelin homolog Scp1 functions with fimbrin to regulate stability and organization of the actin cytoskeleton.

Calponins and transgelins are members of a conserved family of actin-associated proteins widely expressed from yeast to humans. Although a role for calponin in muscle cells has been described, the biochemical activities and in vivo functions of nonmuscle calponins and transgelins are largely unknown. Herein, we have used genetic and biochemical analyses to characterize the budding yeast member of this family, Scp1, which most closely resembles transgelin and contains one calponin homology (CH) domain. We show that Scp1 is a novel component of yeast cortical actin patches and shares in vivo functions and biochemical activities with Sac6/fimbrin, the one other actin patch component that contains CH domains. Purified Scp1 binds directly to filamentous actin, cross-links actin filaments, and stabilizes filaments against disassembly. Sequences in Scp1 sufficient for actin binding and cross-linking reside in its carboxy terminus, outside the CH domain. Overexpression of SCP1 suppresses sac6Delta defects, and deletion of SCP1 enhances sac6Delta defects. Together, these data show that Scp1 and Sac6/fimbrin cooperate to stabilize and organize the yeast actin cytoskeleton.

Actin Cytoskeleton↗

PubMed2002

Electrophoresis using ultra-high voltages.

Optimization of electrophoretic techniques is becoming an increasingly important area of research as microdevices are now routinely adapted for numerous biology and engineering applications. The present work seeks to optimize electrophoresis within microdevices by utilizing ultra-high voltages to increase sample concentration prior to separation. By imaging fluorescently-tagged DNA samples, the effects of both conventional and atypical voltage protocols on DNA migration and separation are readily observed. Experiments illustrate that short periods of high voltage during electrophoretic injection do not destroy the quality of DNA separations, and in fact can enhance sample concentration five-fold. This study presents data that illustrate increases in average resolution, and resolution of longer fragments, obtained from electrophoretic injections utilizing voltages between 85 and 850 V/cm.

DNA↗

PubMed2002

Electrophoretic injection within microdevices.

The flexibility of the microfabricated format creates unique opportunities for study of the electrophoretic process. The present work utilizes digital images to capture the motion of DNA samples during pre-electrophoretic processes. A systematic study of DNA loading and strong sample stacking (sample concentration effects) was performed in order to analyze realistic DNA analysis conditions within microdevices. Using digital imaging and microscopy, DNA sample profiles within the injector were analyzed by deconvolving the geometrical intensity profile into different velocity groups. This analysis illustrates the evolution of molecular separation into distinct migrating populations within the injector itself. The present study performed DNA injections within microfabricated devices imposing run voltages between 85 and 850 V/cm. Data from 3 different offset lengths of a double-T cross-injector, 10 different applied voltages, and 2 different sample preparation protocols are presented.

DNA↗

PubMed2002

High-speed analysis of multiplexed short tandem repeats with an electrophoretic microdevice.

We report the development of a robust and effective method for multiplexed short tandem repeat (STR) analysis within a chip-based microdevice. The method uses a laser-induced fluorescence detection system and simultaneously detects three- and four-color multiplexed polymerase chain reaction (PCR) samples. Analyses of the eight combined DNA index system (CODIS) STR loci were performed in 20 min with single-base-pair resolution ranging from 0.75 to 1. A simultaneous analysis of fifteen loci-ladders and a gender marker Amelogenin based on the PowerPlexTM 16 System was achieved in less than 35 min. The system is capable of repetitive operation and may be extended to high-throughput multilane devices that could be readily interfaced to an automated sample loading system.

Electrophoresis↗